Excess lipid accumulation in non-adipose tissues is associated with insulin resistance, pancreatic a cell apoptosis and heart failure. Previous work has shown that high concentrations of the saturated fatty acid palmitate, but not mono-unsaturated oleate, leads to apoptosis in cultured cells by a mechanism involving the generation of reactive intermediates. Furthermore, palmitate-induced apoptosis can be rescued by co-incubation with oleate. The goal of the present study is to characterize mechanisms of lipotoxicity caused by cellular accumulation of saturated long chain fatty acids, and to gain insight into the fundamental cellular mechanisms through which the common saturated and unsaturated dietary lipids, palmitic and oleic acids, exert their differential effects on cell survival. We will test the hypothesis that cellular proteins participate in channeling of specific long chain fatty acids to different metabolic fates. In Specific Aim 1, we will use Chinese hamster ovary (CHO) cells as a model system in which to study the lipid metabolic and signaling pathways responsible for fatty acid-induced apoptosis. In Specific Aim 2, we will isolate and characterize mutant CHO cell lines that are resistant to palmitate-induced apoptosis. In Specific Aim 3, we will identify the mutations that confer palmitate-resistance in these mutant CHO cells. Through this screen we will identify proteins that participate in channeling palmitic and oleic acids to distinct metabolic fates. The results of these studies will characterize a fundamental aspect of cellular lipid homeostasis. Moreover, these studies will provide new insights into the cytotoxic response to excess lipid accumulation in non-adipose tissues observed in diseases such as diabetes, lipodystrophies, and heart failure.